Inhibitors of agonist-specific desensitization

ABSTRACT

The present invention relates to a method of inhibiting desensitization of a cell to the effects of a compound. The method comprises contacting the cell with an agent capable of inhibiting phosphorylation, by a protein kinase, of a receptor for the compound present on the surface of the cell. The present invention also relates to a method of screening a compound for its ability to inhibit desensitization. The method comprises:  
     i) contacting a receptor specific kinase-containing sample with the compound under conditions such that interaction between receptor specific kinase present in the sample and the compound can occur, and  
     ii) determining the ability of the receptor specific kinase to phosphorylate the receptor for which it is specific.

BACKGROUND OF THE INVENTION

[0001] 1. Technical Field

[0002] The present invention relates, in general, to desensitization,and, in particular, to a method of inhibiting agonist-specificdesensitization, to compounds suitable for use in such a method and topharmaceutical compositions comprising same.

[0003] 2. Background Information

[0004] Desensitization is a general phenomenon which is characterized bya reduced responsiveness following prolonged exposure to a hormone ordrug. Occupancy of a wide variety of hormone and neurotransmitterreceptors by agonists often leads to desensitization, that is, to a lossof receptor responsiveness to subsequent stimulation by agonist. Thisparticular phenomenon is generally termed homologous desensitization.This is in contrast to the heterologous form of desensitization, whichis defined as a loss of receptor responsiveness caused by agonistoccupancy of other receptors.

[0005] Homologous desensitization has been most thoroughly studied forthe β-adrenergic receptor (βAR)-adenylyl cyclase system (Benovic et al(1988) Ann. Rev. Cell Biol. 4:405-428). Homologous desensitization ofβARs is accompanied by receptor phosphorylation (Sibley et al (1985) J.Biol. Chem. 260:3883-3886; Strasser et al (1986) Biochemistry25:1371-1377). A cAMP-independent kinase, termed βAR kinase, has beendescribed that specifically phosphorylates the agonist-occupied forms ofthe β₂-adrenergic receptor (β₂AR) and α₂-adrenergic receptor (Benovic etal (1986) Proc. Natl. Acad. Sci. USA 83:2797-2801; Benovic et al (1987)J. Biol. Chem. 262:17251-17253) as well as light-activated rhodopsin(Benovic et al (1986) Nature (London) 322:867-872). Phosphorylation ofthe β₂AR by βAR kinase may trigger the process of functional uncouplingfrom the stimulatory guanine nucleotide binding protein, G_(s) (Benovicet al (1987) Proc. Natl. Acad. Sci. USA 84:8879-8882).

[0006] While there have been several publications involving attempts atblocking desensitization (Mallorga et al (1980) Proc. Natl. Acad. Sci.USA 77:1341-1345; Shima et al (1983) J. Biol. Chem. 258:2083-2086;Heyworth et al (1984) Biochem. J. 222:189-194; Fano et al (1986) J.Auton. Pharmacol. 6:47-51; DeBernardi et al (1987) Proc. Natl. Acad.Sci. USA 84:2246-2250; and Toews (1987) Mol. Pharmacol. 32:737-742),none of these studies directly demonstrates the inhibition ofagonist-specific desensitization. Moreover, none of the compoundsutilized in these studies directly inhibit the βAR kinase. Theidentification of compounds which specifically inhibit this kinase couldprovide a method of inhibiting desensitization. The inhibition ofdesensitization should result in enhanced and prolonged receptor actionin response to endogenous compounds and drugs. In the case of drugs,this means increased drug efficacy leading to the use of lower drugdoses, and reduced side effects. In addition, it should allow prolongedtreatment with drugs.

SUMMARY OF THE INVENTION

[0007] It is a general object of the invention to provide a method ofblocking agonist-specific desensitization.

[0008] It is a specific object of the invention to provide compoundscapable of inhibiting agonist-specific desensitization.

[0009] It is a further object of the invention to provide apharmaceutical composition comprising as an active ingredient a compoundcapable of blocking agonist-specific desensitization.

[0010] It is another object of the invention to provide a method ofaugmenting the efficacy and duration of treatment of drugs, thecontinued administration of which results in desensitization.

[0011] It is a further object of the invention to provide a method ofrestoring the effectiveness of receptor-mediated responses to endogenouscompounds such as hormones and neurotransmitters.

[0012] Further objects and advantages of the present invention willbecome clear to one skilled in the art from a reading of the descriptionthat follows.

[0013] In one embodiment, the present invention relates to a method ofinhibiting desensitization of a cell to the effects of a compound. Themethod comprises contacting the cell with an agent capable of inhibitingphosphorylation, by a protein kinase, of a receptor for the compoundpresent on the surface of the cell.

[0014] In another embodiment, the present invention relates to a methodof screening a compound for its ability to inhibit desensitization. Themethod comprises:

[0015] i) contacting a receptor specific kinase-containing sample withthe compound under conditions such that interaction between receptorspecific kinase present in the sample and the compound can occur, and

[0016] ii) determining the ability of the receptor specific kinase tophosphorylate the receptor for which it is specific.

[0017] In a further emobodiment, the present invention relates to aninhibitor of β adrenergic receptor kinase. The inhibitor consistsessentially of a peptide corresponding to an intracellular domain of theβ₂ adrenergic receptor.

[0018] In yet another embodiment, the present invention relates to apharmaceutical composition. The composition comprises as an activeingredient the above-described peptide (or other agent) in an amountsufficient to exhibit an inhibitory effect on β adrenergic receptorkinase, together with a pharmaceutically acceptable carrier.

BRIEF DESCRIPTION OF THE DRAWINGS

[0019]FIG. 1. Inhibition of βAR kinase by polyanions. Urea-treated rodouter segments were phosphorylated by βAR kinase in the presence of theindicated concentrations of polyanion. 100% activity corresponds to 11pmol of phosphate incorporated during a 15-min incubation withoutinhibitor. Δ, dextran sulfate; ◯, heparin; , de-N-sulfated heparin; ▴,polyglutamic acid.

[0020]FIG. 2. Lineweaver-Burk plots for the inhibition of βAR kinase byheparin. FIG. 2A, rhodopsin was varied in the assay from 3 to 20 μM. Theheparin concentrations were 0 (◯), 0.21 (), 0.42 (Δ), and 0.63 (▴) μM.FIG. 2B. ATP was varied in the assay from 33 to 100 μM. The heparinconcentrations were 0 (◯), 0.10 (), 0.21 (Δ), and 0.31 (▴) μM.

[0021]FIG. 3. Inhibition of βAR kinase by polycations. Urea-treated rodouter segments were phosphorylated by βAR kinase in the presence of theindicated concentrations of polycation. 100% activity corresponds to 13pmol of phosphate incorporated during a 15-min incubation withoutinhibitor. ▴, polylysine; ◯, spermine; , spermidine.

[0022]FIG. 4. Reversal of heparin inhibition by polycations.Urea-treated rod outer segments were phosphorylated by βAR kinase in thepresence of 1.1 AM heparin and the indicated concentration ofpolycation. 100% activity represents phosphorylation in the absence ofheparin and corresponds to 6 pmol of phosphate incorporated in a 15-minincubation. ▴, polylysine; ◯, spermine; , spermidine.

[0023]FIG. 5. Desensitization of β₂ARs in intact (FIG. 5A) andpermeabilized (FIG. 5B) A431 cells. Cells were incubated for 10 min with(desensitized) () or without (control) (◯) 1 μM (−)-isoproterenol asdetailed. Adenylyl cyclase activities of membranes prepared from thecells were measured in the presence of various concentrations of(−)-isoproterenol and were expressed as percent of the activity in thepresence of 10 mM NaF. Values for EC₅₀ maximal stimulation over basal(E_(max), in percent of activity with 10 mM NaF), and activity in thepresence of 10 mM NaF (=100%, in pmol of cAMP per mg of protein per min)were as follows: for control intact cells they were 200 nM (EC₅₀), 56%(E_(max)), and 84±2 pmol/mg/min (100%); for desensitized intact cellsthey were 300 nM (EC₅₀), 40% (E_(max)), and 70±7 pmol/mg/min (100%) forcontrol permeabilized cells they were 130 nM (ECSO), 62% (E_(max)), and86+2 pmol/mg/min (100%) for desensitized permeabilized cells they were190 nM (EC₅₀), 43% (E_(max)), and 69±3 pmol/mg/min (100%). Data aremeans±SEM of three independent experiments.

[0024]FIG. 6. Time course of β₂AR desensitization in permeabilized A431cells. Permeabilized cells were incubated for the indicated periods oftime with 1 μM (−)-isoproterenol alone (control) or in the presence of100 μM of the peptide β₂AR-(57-71) [β₂(57-71)] or 100 nM heparin.Desensitization was measured as the percent loss of stimulation by 10 μM(−)-isoproterenol of adenylyl cyclase activity in membranes. Data aremeans±SEM of six (control) or three independent experiments.

[0025]FIG. 7. Inhibition by heparin of β₂AR phosphorylation by βARkinase in a reconstituted system (FIG. 7A) and of β₂AR desensitizationin permeabilized A431 cells (FIG. 7B). FIG. 7A: Purified reconstitutedβ₂ARs were phosphorylated by purified βAR kinase with [γ-³²P]ATP in thepresence of various concentrations of heparin. Curve-fitting gave anIC₅₀ value of 6.1±2.0 nM. (Inset) Relevant section of a representativeautoradiogram. Data are means±SEM of three independent experiments. FIG.7B: Permeabilized A431 cells were desensitized by incubation with 1 μM(−)-isoproterenol for 10 min at 37° C. in the presence of variousconcentrations of heparin. Desensitization was measured as the percentloss of stimulation by 10 μM (−)-isoproterenol of adenylyl cyclaseactivity in membranes. Curve-fitting gave an IC₅₀ value of 21+4 nM. Dataare means±SEM of four independent experiments.

[0026]FIG. 8. Phosphorylation of β₂ARs in permeabilized A431 cells.Permeabilized cells were incubated with [γ-³²P]ATP without (CON) or with(ISO) 1 μM (−)-isoproterenol in the absence or presence of 1 μM heparin.β₂ARs were solubilized, purified by affinity chromatography, andelectrophoresed on a 10% sodium dodecyl sulfate polyacrylamide gel, eachlane containing 0.5 pmol of receptor. FIG. 8A: Autoradiogram obtainedafter a 10-day exposure at −70° C. FIG. 8B: Transverse densitometricscan of the autoradiogram. FIG. 8C: An aliquot of purified receptorsfrom cells incubated without (−)-isoproterenol or heparin wasphotoaffinity-labeled with ¹²⁵I-cyanopindolol diazirine in the absence(CON) or presence (ALP) of 10 μM alprenolol, followed by electrophoresison the same gel (60 fmol per lane) and autoradiography. Similar resultswere obtained in two other experiments.

[0027]FIG. 9. Model for the organization of the hamster β₂AR in theplasma membrane. The amino acid sequence and proposed structure of thehamster β₂AR in the plasma membrane (Dixon et al (1986) Nature321:75-79). The synthetic peptides used in this study are bracketed andlabeled: NT—amino terminus; CI, CII, CIII—first, second and thirdintracellular loops; EI, EII—first and second extracellular loops; CT-1,CT-2, CT-3-carboxyl terminus.

[0028]FIG. 10. Time course of phosphorylation of β₂AR and syntheticpeptides by βAR kinase. Reconstituted hamster lung β2-adrenergicreceptor (0.5 pmol) was phosphorylated by βAR kinase (˜30 ng) in thepresence (γ) of 50 μM (−)-isoproterenol. Two synthetic carboxyl terminalpeptides of the hamster lung β₂AR (CT-2 (Δ), CT-3 (γ)) were alsophosphorylated by βAR kinase under identical conditions. Following theincubation period the reactions (20 μl) were stopped by addition of 50μl of SDS sample buffer followed by electro-phoresis on a 10% (β₂AR) or15% (CT-2, CT-3) homogeneous polyacrylamide gel.

[0029]FIG. 11. Kinetic parameters of the βAR kinase for β₂AR.Reconstituted β₂AR (0.05-0.3 μM) was incubated for 15 min at 30° C. withpurified βAR kinase (˜30 ng) in the presence (◯) or absence (γ) of 50 μM(−)-isoproterenol. Reactions were stopped by the addition of SDS samplebuffer before electrophoresis on a 10% SDS polyacrylamide gel.³²P-labeled receptor was determined by cutting and counting the driedgel following autoradiography.

[0030]FIG. 12. Kinetic parameters of the βAR kinase for syntheticpeptides CT-2 and CT-3. The synthetic β₂AR peptides CT-2 (0.6-3.3 mM; Δ)and CT-3 (0.6-4.3 mM; γ) were incubated for 60 min at 30° C. withpurified βAR kinase (˜30 ng). Reactions were stopped by the addition ofSDS urea sample buffer before electrophoresis on a 9% SDS ureapolyacrylamide gel. ³²P-labeled peptide was determined by cutting andcounting the gel following autoradiography.

[0031]FIG. 13. Inhibition of βAR phosphorylation by synthetic peptides.Reconstituted β₂AR (˜0.4 pmol) was incubated for 30 min at 300 withpurified βAR kinase (˜30 ng) in the presence of 50 mM Tris-HC1, pH 7.5,2 mM EDTA, 10 mM NaCl, 5 mM MgCl₂, 5 mM sodium phosphate, 50 μM(−)-isoproterenol and 0.10 mM [γ³²P]ATP (2.0 cpm/fmol). Synthetic β₂ARpeptides were varied from 0 to 1 mM and included CI (Δ), CII (A), CIII-2(◯) and CT-1 (γ). Reactions were stopped by the addition of SDSpolyacrylamide gel. ³²P-labeled receptor was determined by cutting andcounting the dried gel following autoradiography.

DETAILED DESCRIPTION OF THE INVENTION

[0032] The present invention relates to a method of inhibiting thedesensitization of a cell to the effects of a compound (i.e. hormone ordrug), the desensitization resulting from the continued contact of thecell with the compound. The method is based on the discovery that theloss of responsiveness of a cell to a particular compound is due tophosphorylation of the cell surface receptor for that compound.

[0033] In a specific embodiment, the present invention relates to amethod of blocking the agonist specific (homologous) desensitization ofa cell (tissue or organism) that results from prolonged exposure to theagonist. The method comprises contacting the cell (tissue or organism)subject to desensitization with a compound capable of inhibitingphosphorylation of the receptor for a particular agonist.

[0034] Compounds suitable for use in the present invention include thosecapable of inhibiting receptor kinases, for example, specific proteinkinases involved in mediating homologous desensitization of adenylylcyclase-coupled receptors (i.e. βAR kinase). (See also Blackshear (1988)FASEB J. 2:2957; Middleton (1988) Ann. Allergy 61:53; Hunnan (1988)Science 243:500.) Examples of such compounds include polyanions,advantageously, acid mucopolysaccharides such as heparin and dextransulfate. Polycations, for example, polylysine, spermine and spermidine,are also capable of inhibiting the receptor specific kinase βAR kinase.

[0035] Highly receptor specific inhibition can be achieved usingpeptides corresponding to discrete domains of the receptor thephosphorylation of which is sought to be inhibited. For example, theβ₂AR peptides βAR (219-243)(designated CIII-1 below), βAR (56-74)(designated CI below), β₂AR (57-71), β₂AR (57-71; L→A, N→A), and βAR(57-71; K→A, R→A) are relatively potent inhibitors of βAR kinase, whileβ₂AR (97-106) (designated EI below), β₂AR (137-151) (designated CIIbelow), β₂AR (248-268) (designated CIII-2 below), β₂AR (337-355) andβ₂AR (353-381) (designated CT-2 below) and β₂AR (57-71, E→Q), whichpeptides also inhibit βAR kinase, are somewhat less effective. Methodsof identifying receptor peptides capable of exerting an inhibitoryeffect on receptor kinases are set forth in the Examples that follow.The invention includes within its scope receptor peptides, for example,those set forth above.

[0036] The invention also relates to pharmaceutical compositionscomprising as an active ingredient at least one inhibitor of at leastone receptor kinase, together with a pharmaceutical carrier. Suchcompositions can include agents (for example, lipophilic reagents) thatfacilitate transport of the inhibitor across cell walls. Thecompositions can be in dosage unit for (i.e. pill, tablet or injectablesolution, etc.). The amount of active ingredient to be included in sucha composition, and the amount of active ingredient to be administered,can be determined by one skilled in the art without undueexperimentation.

[0037] It will be appreciated that the method of the present inventioncan be used to restore the effectiveness of receptor mediated responsesto endogenous compounds, such as hormones and neurotransmitters.

[0038] In another embodiment, the present invention relates to a methodof screening compounds for their ability to block agonist-specificdesensitization. The method can be practiced using a culture of cellspermeabilized according to known protocols (for example, by treatmentwith digitonin), a cell lysate, or a cell free preparation of receptorkinase(s) (all of which will hereinafter be referred to as “receptorkinase containing sample”). The method comprises contacting the receptorkinase containing sample with a compound to be tested for its ability toinhibit desensitization under conditions such that interaction (i.e.binding) of the receptor kinase present in the sample and the compoundcan occur. The ability of the test compound to inhibit receptor kinaseactivity, and, therefore, block agonist-specific desensitization, isthen determined by assaying for the ability of the kinase tophosphorylate the receptor for which it is specific. Assays forphosphorylation activity are known in the art and are exemplified below.

[0039] The following non-limiting Examples further describe the presentinvention.

Example I Inhibition of βAR Kinase by Polyanions

[0040] Heparin (average M_(r)=4000-6000), de-N-sulfated heparin, dextransulfate (average M_(r)=5000), heparin sulfate, chondroitin sulfate B,chondroitin sulfate C, polyaspartic acid (average M_(r)=11,000),polyglutamic acid (average M_(r)=13,600), inositol hexasulfate, inositolhexaphosphate, pyridoxal phosphate, 2,3-diphosphoglycerate, glucosamine2,6-disulfate, polylysine (average M_(r)=3300), spermine, and spermidinewere purchased from Sigma. Frozen bovine retinas were from Hormel, whilebovine cerebral cortex was obtained from a slaughterhouse.

[0041] βAR kinase was purified from bovine cerebral cortex bymodification of procedure of Benovic et al (J. Biol. Chem. (1987)262:9026-9032). Briefly, 250 g of bovine cerebral cortex werehomogenized with a Brinkmann tissue disrupter and centrifuged (40,000×g,30 min). The supernatant was then precipitated with 13-26% ammoniumsulfate. This material was initially chromatographed on a Ultrogel AcA34column equilibrated with 5 mM Tris-HCl, pH 7.5, 2 mM EDTA, 10 μg/mlleupeptin, 5 μg/ml pepstatin, 10 μg/ml benzamidine, 0.2 mMphenylmethylsulfonyl fluoride (buffer A). The peak activity was dilutedwith an equal volume of buffer A containing 0.04% Triton X-100 beforebeing applied to a DEAE-Sephacel column. Elution was accomplished with a0-80 mM NaC1 gradient in buffer A containing 0.02% Triton X-100. Thepeak activity was then applied to a CM-Fractogel column and eluted witha 0-100 mM NaC1 gradient in buffer A containing 0.02% Triton X-100. Thepurified kinase is stable at 4° C. for several months. For these studiesthe βAR kinase preparations were >75% pure.

[0042] Rod outer segments were prepared from bovine retinas by stepwisesucrose gradient centrifugation (Wilden et al Biochemistry (1982)21:3014-3022). Rhodopsin kinase-free rod outer segments were prepared bytreatment with 5 M urea (Shichi et al J. Biol. Chem. (1978)253:7040-7046) and consisted of −95% rhodopsin, as assessed by CoomassieBlue staining of sodium dodecyl sulfate-polyacrylamide gels.

[0043] Urea-treated rod outer segments were phosphorylated with βARkinase as follows. Urea-treated rod outer segments (˜4 μg of rhodopsin)were incubated with βAR kinase (˜0.1 μg) for 10-15 min at 30° C. in thepresence of 40 mM Tris-HC1, pH 7.4, 10 mM NaC1, 5 mM MgCl₂, 1.5 mM EDTA,and 65 μM ATP (˜1 cpm/fmol) (total reaction volume was 40 μ1). Reactionswere stopped by the addition of 40 μ1 of sodium dodecyl sulfate samplebuffer (see below) followed by sodium dodecyl sulfate-polyacrylamide gelelectrophoresis. After autoradiography, the phosphorylated rhodopsinbands were excised and counted.

[0044] βAR kinase phosphorylates the β₂AR, the α₂-adrenergic receptor,and rhodopsin in a stimulus-dependent fashion. Rhodopsin was utilized asthe substrate in these experiments because of its ease of isolation.However, the inhibitory effects of polyanions observed with βARkinase-catalyzed phosphorylation of rhodopsin have also been observedusing reconstituted β₂AR as the substrate.

[0045] Sodium dodecyl sulfate-polyacrylamide gel electrophoresis wasperformed by the method of Laemmli (Nature (1970) 227:680-685) using 10%homogeneous polyacrylamide slab gels. Sample buffer contained 8% sodiumdodecyl sulfate, 10% glycerol, 5% 3-mercaptoethanol, 25 mM Tris-HC1, pH6.5, and 0.003% bromphenol blue. After electrophoresis, gels were driedwith a Bio-Rad gel dryer prior to autoradiography.

[0046]FIG. 1 demonstrates that a number of polyanions are potentinhibitors of βAR kinase-catalyzed phosphorylation of rhodopsin. Inparticular, heparin and dextran sulfate appear to be the most potent.De-N-sulfated heparin is ˜8-fold weaker suggesting some importance ofthe N-sulfate. It is interesting to note that the inhibition by heparin,dextran sulfate, and de-N-sulfated heparin appears to involve acooperative interaction of the polyanion with βAR kinase, as the data inFIG. 1 yield a Hill coefficient of ˜2. Several other related acidmucopolysaccharides, such as heparan sulfate and chondroitin sulfates Band C, are significantly less potent than heparin (Table I). The IC₅₀values of these compounds as well as those of a number of other anioniccompounds are shown in Table I. TABLE I Effect of polvanions on theactivity of βAR kinase Phosphorylation of rhodopsin by βAR kinase in thepresence of increasing concentrations of several polyanions was assessedas described above. The IC₅₀ is the concentration of compound which gave50% inhibition and is presented as the mean ± S.E. with the number ofdeterminations given in parenthesis. ND, molar concentration cannot beaccurately determined. IC₅₀ Compound μg/ml μM Heparan 0.77 ± 0.10 (5)0.15 De-N-sulfated heparin 6.0 ± 2.4 (3) ND Heparan sulfate 1.8 NDChondroitin sulfate B 25 ND Chondroitin sulfate C 6 ND Dextran sulfate0.76 ± 0.21 (3) 0.15 Polyaspartic acid 14 1.3 Polyglutamic acid 27 ± 2(2) 2.0 Inositol hexasulfate 12 ± 0.5 (2) 13.5 Inositol hexaphosphate3325 3600 Pyridoxal phosphate 222 900 2,3-Diphosphoglycerate 838 1100Glucosamine 2,6-disulfate 2800 7300

[0047] It is interesting that inositol hexasulfate is ˜270 times morepotent than inositol hexaphosphate. This suggests again that the sulfatemoiety and not just the anionic character of the molecule is animportant determinant in the inhibition of βAR kinase. Several othercompounds such as pyridoxal phosphate, 2,3-diphosphoglycerate, andglucosamine 2,6-disulfate inhibited in the millimolar range. Similarresults are observed when the βAR is used as the substrate with heparinagain being the most potent inhibitor (IC₅₀-0.03 μM, data not shown).

[0048] Since heparin is the most potent inhibitor, the kinetics ofheparin inhibition of βAR kinase were further characterized.

[0049]FIG. 2A shows a Lineweaver-Burk plot of data where the rhodopsinconcentration is varied in the presence of several differentconcentrations of heparin. In the absence of heparin, a K_(m)=5.3 μM wasobtained. Heparin appears to be a competitive inhibitor with respect torhodopsin. A replot of these data (slope versus heparin concentration,not shown) yields a K_(i)˜0.5 μg/ml (˜0.10 μM). FIG. 2B shows a similarplot when the ATP concentration is varied in the presence of severaldifferent concentrations of heparin. These data suggest that heparin isa mixed type inhibitor with respect to ATP.

[0050] Polyanions are known to inhibit casein kinase II, whereaspolycations, such as spermine, spermidine, and putrescine, are able toactivate casein kinase II at low Mg²⁺ or at 50-100 mM KC1 concentrations(Hathaway et al (1982) Curr. Top. Cell. Regul. 21:101-127). In contrast,spermine and spermidine are weak inhibitors of βAR kinase (FIG. 3).Polylysine is more potent with an IC₅₀˜69 μM. Similar results areobserved at low Mg²⁺ concentrations, as well as when 50 mM KC1 isincluded in the incubation.

[0051] At lower concentrations, polycations are able to partiallyreverse the inhibition of βAR kinase by heparin (FIG. 4). Polylysine ismost effective with an EC₅₀˜2.8 μM and a recovery of ˜100% of theinitial uninhibited activity. Spermine and spermidine also reverse theinhibition with EC₅₀ values of 40 and 630 μM and recoveries of ˜68 and57% activity, respectively. At higher concentrations these polycationsare again inhibitory to the system. The mechanism of this reversal mostlikely represents a direct interaction of the polycation with heparin.

[0052] In summary, these results indicate that polyaniqns, in particularheparin and dextran sulfate, are potent inhibitors of the β-adrenergicreceptor kinase. Polycations such as polylysine, spermine, andspermidine also inhibit βAR kinase activity. Polycations are also ableto reverse the inhibitory effects of heparin.

Example II Inhibition of βAR Kinase Prevents Rapid HomologousDesensitization of β₂-ARs

[0053] [α-³²P]ATP, [γ-³²P]ATP, [3H]cAMP, ¹²⁵I-labeled cyanopindolol(¹²⁵I-cyanopindolol), and ¹²⁵I-cyanopindolol diazirine were obtainedfrom New England Nuclear; heparin (H-3125 from porcine mucosa), fromSigma. 1-5(isoquinoline sulfonyl)-2-methylpiperazine (designated H-7),from Calbiochem; and digitonin, from Gallard Schlessinger. The peptidecorresponding to residues 1-24 of the heat-stable inhibitor ofcAMP-dependent protein kinase [PKI-(1-24) tetracosapeptide: Scott et alProc. Natl. Acad. Sci. USA (1986) 83:1613-1616] and the β₂AR-(57-71)pentadecapeptide(Ala-Ile-Ala-Lys-Phe-Glu-Arg-Leu-Gln-Thr-Val-Thr-Asn-Tyr-Phe; Kobilka etal Proc. Natl. Acad. Sci USA (1987) 84:46-50) and β₂AR-(59-69)undecapeptide (Ala-Lys-Phe-Glu-Arg-Leu-Gln-Thr-Val-Thr-Asn) werechemically synthesized.

[0054] Purified β₂AR was phosphorylated by purified βAR kinase asfollows. β₂ARs from hamster lung were purified by affinitychromatography and HPLC to >95% homogeneity (Benovic et al (1984)Biochemistry 23:4510-4518). βAR kinase was purified from bovine cerebralcortex by precipitation with ammonium sulfate, followed bychromatography on Ultrogel AcA34, DEAE-Sephacel, and CM-Fractogelto >75% purity as described (see Example I and Benovic et al (1987) J.Biol. Chem. 262:9026-9032). Purified β₂ARs were inserted intophosphatidylcholine vesicles by chromatography on Extracti-gel, followedby polyethylene glycol treatment and centrifugation at 280,000×g for 90min (Cerione et-al (1984) Biochemistry 23:4519-4525). Phosphorylation ofthe reconstituted receptors by purified βAR kinase was done in thepresence of 50 μM (−)-isoproterenol as described (Benovic et al (1987)J. Biol. Chem. 262:9026-9032). Subsequently, the samples were subjectedto sodium dodecyl sulfate polyacrylamide gel electrophoresis on 10% gels(Laemmli (1970) Nature 227:680-685). The phosphorylated β₂ARs werevisualized by autoradiography, and the corresponding bands were cut outand their content of ³²p quantified.

[0055] Permeabilization of cells was accomplished as follows. Humanepidermoid carcinoma A431 cells were grown to about 95% confluence inDulbecco's modified Eagle's medium supplemented with 10% fetal calfserum. Cells were harvested with collagenase, washed three times withcalcium-free phosphate-buffered saline (PBS), then washed twice in 150mM potassium glutamate/10 mM Hepes/5 mM EGTA/7 mM MgC1₂, pH 7.1 (KGbuffer), and finally resuspended in KG buffer supplemented with 5 mMglucose and 2 mM ATP (KG-A buffer) at a density of 4×10⁷ cells per ml.

[0056] The concentration of digitonin required to permeabilize the cellswas found to vary considerably with cell density. At 4×10⁷ cells per ml,about 0.015% digitonin was necessary to achieve permeabilization of >95%cells as assessed by staining with trypan blue. In all experiments,digitonin was added stepwise until >95% of the cells were trypan bluepositive.

[0057] Desensitization of β₂ARs in A431 cells was effected as follows.Permeabilized cells in KG-A buffer (or, for controls, intact cells inPBS) were incubated with or without 1 βM (−)-isoproterenol for 10 min orthe indicated times (4×10⁷ cells per ml). The incubation was terminatedby addition of 10 vol of ice-cold KG buffer (or PBS, respectively),followed by centrifugation at 1000×g for 5 min. After three identicalwashes, cells were disrupted with a Polytron homogenizer in 5 mMTris-HCl, pH 7.4/2 mM EDTA. Crude membranes were prepared by spinningthe supernatant after low-speed centrifugation (1000×g for 5 min) at40,000×g for 20 min.

[0058] Adenylyl cyclase activity in the membranes was determined asdescribed by Salomon et al (Anal. Biochem. (1974) 58:541-548). The freeMg²⁺ concentration in the assay was 4 mM. The incubation lasted for 20min at 37° C.; accumulation of [³² P]cAMP was linear over this timeperiod.

[0059] β₂ARs in permeabilized A431 cells were phosphorylated as follows.Permeabilized cells (4×10⁸) in 10 ml of KG-A buffer containing 1 mCi (1Ci=37 GBq) of [γ-⁻³²p]ATP were incubated with or without 1 μM(−)-isoproterenol (and other compounds as indicated) for 10 min at 37°C. All buffers used in these experiments contained 5 μg of soybeantryspin inhibitor, 5 μg of leupeptin, and 10 μg of benzamidine per mland 0.1 mM phenylmethylsulfonyl fluoride. The reaction was terminated byaddition of 30 ml of 150 mM potassium glutamate/5 mM EDTA/10 mM sodiumphosphate, pH 7.1, at 0° C. and centrifugation at 1000×g for 5 min.After three identical washing steps, crude membranes were prepared asabove. The membranes were solubilized with 2% digitonin, and β₂ARs werepurified by affinity chromatography with alprenolol-Sepharose (Benovicet al (1984) Biochemistry 23:4510-4518). Equal amounts of β₂ARs fromeach sample (γ0.5 pmol, determined by radioligand binding using¹²⁵I-cyanopindolol) were subjected to sodium dodecyl sulfatepolyacrylamide gel electrophoresis as described above. Gels were fixedin 40% (vol/vol) methanol and 15% (vol/vol) acetic acid and dried priorto autoradiography.

[0060] In addition, purified β₂ARs in these experiments were identifiedby photoaffinity labeling with 125I-cyanopindolol diazirine. Aliquots ofthe samples (γ60 fmol of β₂ARs) were desalted over Sephadex G-50 andincubated for 2 hr at 20° C. with 200 pM of the ligand with or without10 μM alprenolol. Free ligand was removed by desalting as above, and thesamples were irradiated with UV light for 3 min. The samples were loadedon the same gel as the samples described above.

[0061] Concentration-response curves were analyzed by nonlinearcurve-fitting to the Hill equation as described (Lohse et al (1986) Mol.Pharmacol. 30:403-409). Adenylyl cyclase activity was expressed as thepercentage of the activity in the presence of 10 mM NaF. Since NaFstimulates adenylyl cyclase via the stimulatory guanine nucleotidebinding protein, G_(s), this normalizes for effects that occur at thelevel of G_(s) or the cyclase (i.e., which effects representheterologous desensitization). Desensitization was assessed by measuringthe loss of maximal stimulation by isoproterenol [determined with 10 AM(−)-isoproterenol] and was calculated as [1-stimulation(desensitized)/stimulation (control)]×100. For example, a decrease from60% to 40% of NaF-stimulated activity corresponds to a desensitizationof [1-(40/60)]×100=33%.

[0062] A variety of permeabilization techniques were tested for theirability to provide constant and reproducible access to the cytosol whileleaving homologous desensitization unaltered. These includedscrape-loading (McNeil et al (1984) J. Cell Biol. 98:1556-1564),electro-permeabilization (Knight et al (1986) Biochem. J. 234:497-506),and permeabilization with staphylococcal a toxin (Fussle et al (1981) J.Cell Biol. 91:83-94) and different detergents. Permeabilization withdigitonin gave the most consistent results and was used for all futureexperiments.

[0063]FIG. 5 shows that permeabilization of A431 cells with digitonindid not change the pattern of homologous desensitization toisoproterenol (1 μM). Isoproterenol-induced stimulation of adenylylcyclase in membranes from cells incubated with 1 μM isoproterenol for 10min at 37° C. was reduced in both potency and maximal effect as comparedwith membranes of control cells. In both intact and permeabilized cells,the extent of maximal stimulation over basal activity was reduced by thepretreatment from γ60% to γ40% of NaF-stimulated activity, correspondingto a desensitization of γ30%.

[0064]FIG. 6 shows the time course of desensitization in permeabilizedcells in the absence or presence of two inhibitors of βAR kinase,heparin (100 nM) and a peptide corresponding to the first intracellularloop of the human β₂AR [β₂AR-(57-71), 100 μM]. In the absence ofinhibitors, desensitization occurred with a half-time of <5 min.β₂AR-(57-71) markedly slowed the rate of desensitization, anddesensitization was almost completely abolished by 100 nM heparin.Neither compound affected desensitization in intact cells (data nowshown).

[0065] It has been shown that heparin and its analogues may alterstimulation of adenylyl cyclase at micromolar concentrations (Amsterdamet al (1978) Biochim. Biophys. Acta 544:273-283). To ascertain that theinhibition of desensitization by heparin was not due to a reducedformation of cAMP, the accumulation of [³²P]cAMP from [γ-³²P]ATP addedto permeabilized cells was determined. The presence of up to 1 μMheparin did not reduce [³²P]cAMP formation either in the presence or theabsence of isoproterenol. Thus, the data indicate that compounds thatinhibit βAR kinase slow down or suppress homologous desensitization.

[0066] To show that these effects are indeed due to inhibition of βARkinase, the concentration dependence for inhibition of βAR kinaseactivity in a reconstituted system was compared with that for inhibitionof β₂AR desensitization by heparin (FIG. 7). Heparin inhibitedphosphorylation of pure reconstituted β₂ARs by βAR kinase with an IC₅₀value of 6 nM and caused almost complete inhibition at 30-100 nM (FIG.7A). Desensitization of β₂ARs in permeabilized A431 cells was inhibitedby heparin with an IC₅₀ value of 20 nM, and inhibition was virtuallycomplete at concentrations above 100 nM. Thus, theconcentration-response curves for the two effects are similar.

[0067] If the inhibition of desensitization by heparin is in fact causedby inhibition of βAR kinase, then it would be expected that heparinwould reduce the isoproterenol-induced phosphorylation of β₂ARs inpermeabilized cells. Therefore, permeabilized A431 cells were incubatedwith [γ-³²p]ATP with or without 1 AM isoproterenol in the absence orpresence of 1 μM heparin. Incubation time (10 min) and conditions werethe same as in the desensitization experiments reported in FIG. 7. Inthe absence of heparin, isoproterenol caused a 5-fold increase in thephosphorylation of the β₂ARs (FIG. 8). Heparin (1 μM) markedly reducedthis increase while not affecting basal phosphorylation. Quantitation byscanning of the autoradiograms showed that heparin reduced theisoproterenol-induced phosphorylation by 50-80% in three experiments(FIG. 8B). Although some receptor degradation appeared to occur (asevidenced by a band of lower molecular weight), photoaffinity labelingwith ¹²⁵I-cyanopindolol diazirine confirmed that the purifiedphosphorylated bands represent β₂ARs (FIG. 8C). Thus, inhibition of β₂ARdesensitization by heparin is paralleled by an inhibition ofisoproterenol-induced receptor phosphorylation.

[0068] Finally, the effects of other kinase inhibitors and of analoguesof the β₂AR peptide both on phosphorylation of reconstituted β₂ARs byβAR kinase and on desensitization in permeabilized A431 cells werecompared (Table II). The peptide PKI-(1-24), which inhibitscAMP-dependent protein kinase with a K_(i) value of 5×10⁻⁹ M (Scott etal (1986) Proc. Natl. Acad. Sci. USA 83:1613-1615), had no effect atconcentrations up to 1 μM on either βAR kinase activity ordesensitization. H7, an inhibitor of protein kinase C and cyclicnucleotide-dependent protein kinases with K_(i) values of about 5 μM(Hidaka et al (1984) Biochemistry 23:5036-5041), also did not affecteither βAR kinase activity or desensitization at 1 μM and 10 μM. At 100μM it caused γ20% inhibition of both processes. TABLE II Inhibition ofβAR kinase and of desensitization by kinase inhibitors and peptides ofthe β₂AR Inhibition Inhibition Concentration of of Compound μM βARkinase, % Desensitization, % Kinase inhibitors Heparin 0.1 96 ± 1  102 ±16  PKI-(1-24) 1 0 ± 9  0 ± 12 H7 1 3 ± 4 0 ± 4 10 2 ± 7 7 ± 5 100 19 ±6  22 ± 14 β₂AR peptides β₂AR-(57-71) 100 80 ± 13 52 ± 12 β₂AR-(59-69)100  0 ± 6  3 ± 12

[0069] While the 15-amino acid peptide representing the u firstintracellular loop of the human β₂AR at a concentration of 100 μM causedsignificant inhibition of βAR kinase activity, the central 11-amino acidsegment was virtually inactive in this respect. In parallel, the15-amino acid peptide markedly inhibited desensitization (see also FIG.6), whereas the shorter peptide did not affect it. These data confirm acorrelation between inhibition of βAR kinase and inhibition ofhomologous desensitization.

Example III Synthetic Peptides of the Hamster β₂AR as Substrates andInhibitors of the βAR Kinase

[0070] Peptides were synthesized by tBOC chemistry on an AppliedBiosystems 430A Synthesizer. Peptides were deblocked by HF treatment andwere purified by reverse phase high performance liquid chromatography ona C18 column using a 0-50% acetonitrile gradient.

[0071] The β2-adrenergic receptor from hamster lung was purified toapparent homogeneity by sequential affinity and high performance liquidchromatography as described (Benovic et al (1984) Biochemistry23:4510-4518). The purified receptor was reinserted intophosphatidylcholine vesicles as previously described (Cerione et al(1983) Nature 306:562-566). The protein-lipid pellets were resuspendedin 20 mM Tris-HCl, pH 7.2, 2 mM EDTA and used as a substrate for the βARkinase.

[0072] βAR kinase was purified from bovine cerebral cortex bymodification of a previously described procedure (Benovic et al (1987)J. Biol. Chem. 262:9026-9032). Briefly, 250 g of bovine cortex washomogenized and the resulting high speed supernatant fraction wasprecipitated with 13-26% ammonium sulfate. This material was initiallychromatographed on an Ultrogel AcA34 column equilibrated with 5 mMTris-HCl, pH 7.5, 2 mM EDTA, 5 μg/ml leupeptin, 5 μg/ml pepstatin, 15pg/ml benzamidine, 0.2 mM phenylmethyl sulfonyl fluoride (buffer A). Thepeak activity was diluted 1:1 with buffer A containing 0.02% tritonX-100 and applied to a DEAE Sephacel column. Elution was accomplishedwith a 0-80 mM NaCl gradient in buffer A containing triton X-100. Thepeak activity was then applied directly to a CM Fractogel column andeluted with a 0-100 mM NaCl gradient in buffer A containing tritonX-100. The purified kinase was stored at 4° C.

[0073] Phosphorylation of βAR was accomplished by incubatingreconstituted βAR (0.5-5 pmol/incubation) with βAR kinase (10-50 ng) ina buffer containing 20 mM Tris-HCl, pH 7.5, 2 mM EDTA, 10 mM NaCl, 5 mMMgCl2, 5 mM sodium phosphate, 0.5 mM ascorbic acid, 0.15 mM [γ-³²P]ATP(1-5 cpm/fmol) at 30° C. for the indicated time (see Figures indicatedbelow and corresponding description). Some incubations also contained 50μM (−)-isoproterenol. Incubations were stopped by the addition of 50 μlof SDS sample buffer followed by electrophoresis on 10% homogeneouspolyacrylamide gels. Phosphorylated βAR was visualized byautoradiography and the corresponding bands were excised and counted todetermine the pmol of phosphate incorporated.

[0074] Synthetic βAR peptides were phosphorylated as follows by βARkinase. Synthetic peptides derived from the hamster lung β₂AR wereincubated with βAR kinase (50-100 ng) in a buffer containing 50 mMTris-HCl, pH 7.5, 2 mM EDTA, 10 mM NaCl, 5 mM MgCl₂, 1 mMdithiothreitol, 0.10 mM [γ-32P]ATP (1-5 cpm/fmol) at 30° C. for theindicated time (see Figures indicated below and correspondingdescriptions). The peptide concentrations varied from 0.5-6 mM. Peptideswere separated from free ATP by one of several methods. In mostexperiments the reactions were stopped by the addition of 50 μl of SDSsample buffer or SDS urea sample buffer followed by electrophoresis oneither a 15% homogeneous polyacrylamide gel or a 9% polyacrylamide gelcontaining 6.5 urea (see below). Following autoradiography thephosphorylated peptides were excised and counted to determine the amountof phosphate incorporated. In some experiments, samples were separatedby reverse phase high performance liquid chromatography on a C18 column(Kuenzel et al (1985) Proc. Natl. Acad. Sci. USA 82:737-741). Followinginjection of the sample the column was washed with 30 ml of buffer (0.1M sodium phosphate, pH 6.5, 0.1 M NaCl) before elution with a lineargradient from 0 to 50% acetonitrile. An alternative method involveddirect application of the sample to phosphocellulose paper followed byextensive washing in 75 mM H₃PO₄ (Cook et al (1982) Biochemistry21:5794). While this procedure gave comparable results for the CT-2peptide, the CT-3 peptide did not appear to bind appreciably to thephosphocellulose paper.

[0075] The inhibition of substrate phosphorylation by βAR kinase in thepresence of synthetic peptides was assayed as follows. Reconstituted βAR(0.2-1 pmol/incubation) was incubated with βAR kinase (10-30 ng) in abuffer containing 50 mM Tris-HCl, pH 7.5, 2 mM EDTA, 10 mM NaCl, 5 mMMgCl₂, 5 mM sodium phosphate, 50 μM (−)-isoproterenol, 0.10 mM[γ-³²PJATP (1-3 cpm/fmol) at 30° C. for 30 min. Synthetic β₂AR peptideswere varied in concentration from 0 to 2 mM. Reactions were quenched bythe addition of SDS sample buffer followed by gel electrophoresis. Insome experiments urea treated rod outer segments (Wilden et al (1982)Biochemistry 21:3014-3022; Shichi et al (1978) J. Biol. Chem.253:7040-7046) were used as the substrate for βAR kinase. To furtherdefine the specificity of the peptide inhibition, phosvitin, casein (seeExample I) and the synthetic peptides CT-2 and CT-3 were used assubstrates for βAR kinase.

[0076] SDS polyacrylamide gel electrophoresis was performed by themethod of Laemmli (Nature (1970) 227:660-685) using 10 or 15%homogeneous slab gels. SDS sample buffer contained 8% SDS, 10% glycerol,5% β-mercaptoethanol, 25 mM Tris-HCl, pH 6.5 and 0.003% bromphenol blue.SDS urea polyacrylamide gel electrophoresis was carried out using 6.5 Murea, 0.1% SDS, 100 mM H₃PO₄, pH 6.8 with Tris-HCl and 9% acrylamide(Swank et al (1971) Anal. Biochem. 39:462). SDS urea sample buffercontained 1% SDS, 8M urea, 10 mM H₃PO₄, pH 6.8 with Tris-HCl, 1%8-mercaptoethanol and 0.003% bromphenol blue.

[0077]FIG. 9 presents the amino acid sequence and proposed topology ofthe hamster lung β₂AR. The synthetic peptides utilized in this study arehighlighted and encompass the amino and carboxyl termini as well as thefirst and second extracellular and first, second and third intracellularloops.

[0078] Initial studies focused on determining whether any of these 10peptides could serve as substrates for βAR kinase. The results shown inFIG. 10 demonstrate that two of the carboxyl terminal peptides (CT-2 andCT-3) are phosphorylated by βAR kinase. These peptides are, however,much poorer substrates than the agonist-occupied β₂AR even when presentat a 100,000-fold higher concentration. Phosphoamino acid analysis ofthe phosphorylated peptides reveals that CT-2 contains solelyphosphoserine while CT-3 contains predominately phosphoserine with somephosphothreonine (data not shown). None of the other peptides tested arephosphorylated by βAR kinase, however, two of these peptides (CIII-2 andCT-1), which contain a consensus sequence for cAMP dependent proteinkinase phosphorylation, serve as substrates for protein kinase A.

[0079] The kinetics of β₂AR phosphorylation by βAR kinase in thepresence or absence of the β-agonist isoproterenol are shown in FIG. 11.It is evident that the major agonist-promoted difference in thephosphorylation of the receptor is in the Vmax, with theagonist-occupied receptor having an ˜6 fold higher Vmax. (37 vs 6.6 nmolPi/min/mg, Table III). In contrast, the Km of the receptor varies only1.8-fold (0.16 vs 0.29 AM). Overall, the agonist-occupied receptor is an˜10-fold better substrate than the unoccupied receptor as assessed bythe Vmax/Km ratio. In contrast, the phosphorylation of the syntheticβ₂AR peptides by βAR kinase have strikingly different kinetics (FIG.12). The carboxyl peptide CT-2 is phosphorylated by βAR kinase with a Km˜5 mM and a Vmax ˜2.8 nmol Pi/min/mg while CT-3 has a Km ˜8 mM and aVmax ˜2.4. A comparison of the Vmax/Km ratios demonstrates that theagonist-occupied receptor is an ˜10⁶-fold better substrate than thecarboxyl peptides (Table III). The major difference between thesubstrates (receptor vs. peptides) is in the Km obtained with a32,000-50,000 fold difference. TABLE III βAR kinase phosphorylation ofβ₂AR and synthetic β₂AR peptides Km Vmax SUBSTRATE (μM) (nmol/min/mg)Vmax/Km Ratio β₂AR + ISO 0.16 37 231 1 β₂AR 0.29 6.6  23 10⁻¹ CT-2 52002.8 5.4 × 10⁻⁴ 2.3 × 10⁻⁶ CT-3 7900 2.4 3.0 × 10⁻⁴ 1.3 × 10⁻⁶

[0080] Overall, these results demonstrate that the intact β₂AR serves asa much better substrate for βAR kinase than the peptides suggesting thatthe secondary or tertiary structure of the receptor is important forkinase recognition. These results also indicate that βAR kinaserecognizes other regions of the receptor in addition to thephosphorylation sites at the carboxyl terminus.

[0081] To address whether other regions of the β₂AR might interact withβAR kinase, the synthetic peptides were tested as potential inhibitorsof β₂AR phosphorylation. As shown in FIG. 13 a number of syntheticpeptides are potent inhibitors of βAR phosphorylation by βAR kinase.These include the first intracellular loop (C-I) which has an IC₅₀˜40 μMas well as the second and third intracellular loops (CII, CIII-1 andCIII-2) and the carboxyl terminus (CT-1 and CT-2) which have IC₅₀sranging from 70-320 μM (Table IV). Several peptides do not inhibit thephosphorylation when tested at a concentration of ˜0.5 mM (NT, E-II, andCT-3). These results indicate that βAR kinase may interact with multipleregions of the receptor, perhaps explaining the ˜30,000-fold differencein Km between the intact receptor and synthetic peptides forphosphorylation by βAR kinase (Table III). TABLE IV Synthetic peptidesof the hamster β2AR as inhibitors of β₂AR phosphorylation by βAR kinaseIC₅₀ Peptide (μM) NT (17-32) ND CI (56-74)  40 EI (97-106) 300 CII(137-151) 240 EII (177-190) ND CIII-1 (219-243)  76 CIII-2 (248-268) 208CT-1 (337-355) 320 CT-2 (353-381) 142 CT-3 (396-418) ND

[0082] Since the first intracellular loop β₂AR peptide was the mostpotent inhibitor of β₂AR phosphorylation by βAR kinise the specificityof this inhibition was studied further. As the length of the peptidedecreases the ability to inhibit βAR kinase also decreases (Table V). Inparticular, shortening of the peptide from 15 to 11 amino acids resultsin a dramatic loss of the inhibition (IC₅₀ increases from 62 to 2600μM). This indicates that one or more of the four amino acids removed(A,I,Y,F) is critical for the inhibition.

[0083] Several mutant peptides with amino acid substitutions have alsobeen synthesized. A peptide with Leu⁶⁴ and Asn⁶⁹ both changed to alaninedoes not appear to affect the inhibition. Converting Lys⁶⁰ and Arg⁶³ toalanine also does not significantly affect the inhibition. In contrast,a peptide which has Glu⁶² changed to glutamine has significantly loweraffinity as an inhibitor (IC₅₀ from 62 to 280 μM) suggesting that theglutamic acid is important in kinase interaction. Also shown in Table Vare results from studies with peptides derived from the firstintracellular loops of the β1-adrenergic receptor, the α2-adrenergicreceptor and rhodopsin. It is evident that these peptides do notsignificantly inhibit β₂AR phosphorylation by βAR kinase. Interestinglythe β₁AR peptide differs by only four amino acids (out of 15) from theβ₂AR peptide. However, one of these amino acids is Glu⁶² which appearsto be important for inhibition. TABLE V Comparison of firstintracellular loop peptides as inhibitors of βAR kinase IC₅₀ PeptideSequence (μN) β2AR (56-74) TAIAKFERLQTVTNYFITS 40 β2AR (57-71)AIAXFERLQTVTNYF 62 β2AR (59-69) AKFERIJQTVTN 1600 β2AR (60-66) KFERLQT2600 β2AR (57-71, L→A, N→A) AIAKFERAQTVTAYF 43 β2AR (57-71, K→A, R→A)AIAAFEALQTVTNYF 88 β2AR (57-71, E→Q) AIAIKFQRLQTVTNYF 280 β1ARAIAKTPRLQTLTNLF 700 α2AR AVFTSRALKAPQNLF >1000 Rhodopsin VTVQHKKLRTPLNYI>1000

[0084] Further confirming the specificity of the peptide inhibition isthe observation that the peptides failed to inhibit βAR kinase mediatedphosphorylation of non-receptor substrates such as casein and phosvitin.Moreover, the first, second and third intracellular loop peptides didnot inhibit βAR kinase phosphorylation of the carboxyl terminal βARpeptides.

[0085] For purposes of completing this disclosure, the entire contentsof all publications cited hereinabove, are hereby incorporated byreference.

[0086] It will be clear to one skilled in the art to which the presentinvention relates from a reading of the above disclosure that certainchanges in form and detail can be made without departing from the truescope of the invention.

What is claimed is:
 1. A method of inhibiting desensitization of a cellto the effects of a compound comprising contacting said cell with anagent capable of inhibiting phosphorylation, by a protein kinase, of areceptor for said compound present on the surface of said cell.
 2. Themethod according to claim 1 wherein said desensitization is homologousdesensitization.
 3. The method according to claim 1 wherein saidcompound is an endogenous compound.
 4. The method according to claim 1wherein said compound is a drug.
 5. The method according to claim 1wherein said receptors are β₂ adrenergic receptors.
 6. The methodaccording to claim 1 wherein said protein kinase is β adrenergicreceptor kinase.
 7. The method according to claim 1 wherein said cell isa human cell.
 8. A method of screening a compound for its ability toinhibit desensitization comprising: i) contacting a receptor specifickinase-containing sample with said compound under conditions such thatinteraction between receptor specific kinase present in said sample andsaid compound can occur, and ii) determining the ability of saidreceptor specific kinase to phosphorylate the receptor for which it isspecific.
 9. The method according to claim 8 wherein said receptorspecific kinase is β adrenergic receptor kinase.
 10. The methodaccording to claim 8 wherein said desensitization is homologousdesensitization.
 11. The method according to claim 8 wherein saidreceptor specific kinase containing sample is derived from a humansource.
 12. An inhibitor of β adrenergic receptor kinase consistingessentially of a peptide corresponding to an intracellular domain of theβ₂adrenergic receptor.
 13. The inhibitor according to claim 12 whereinsaid peptide is selected from the group consisting residues 56-74,residues 57-71 and residues 219-234.
 14. A pharmaceutical compositioncomprising as an active ingredient a peptide according to claim 12 in anamount sufficient to exhibit an inhibitory effect on β adrenergicreceptor kinase, together with a pharmaceutically acceptable carrier.